Copper is an essential trace element with a critical role in the biochemistry of cellular respiration, antioxidant defense and iron homeostasis. The liver is the central organ of copper homeostasis in humans and the long-term objective of these studies is to define the role of copper in pediatric liver disease. While previous studies have revealed a central role for the copper chaperone Atox1 and the Wilson disease ATPase (ATP7b) in hepatic copper metabolism, the mechanisms within the hepatocyte secretory pathway that lead to excretion of this metal at the canalicular membrane remain poorly understood. Most recently, a direct interaction has been demonstrated between ATP7b and Murr1, the human homologue of the Bedlington terrier copper toxicosis gene product, providing biochemical evidence in support of a role for Mutt1 in the hepatobiliary pathophysiology of copper excretion. The specific aims of this proposal are intended to elucidate the role of Murr1 and ATP7b in this process. Structure/function studies will be accomplished using site-directed mutagenesis to delineate the biochemical details of Murr1 interaction with ATP7b. The pathophysiology of hepatic copper toxicosis in patients with missense mutations in Murr1 will be elucidated by examining copper homeostasis and ATP7b function in mice transgenic for these mutations as well as mice with a targeted germline deletion of the Murr1 gene. The functional role of Murr1 in copper excretion will be determined by isolation and characterization of specific interacting proteins present in Murr1 heterooligomeric complexes formed upon interaction with ATP7b. Finally, the role of the carboxyl terminus of ATP7b in copper-mediated trafficking and excretion in hepatocytes will be defined using in vitro expression cloning to characterize the proteins interacting with this domain. Taken together the results of these studies will permit new insights into the hepatobiliary pathophysiology of excretory events within the liver and may allow for novel therapeutic approaches to prevent or ameliorate hepatic injury in a number of pediatric liver diseases.